Re: 식이 질산염 --＞ 아질산염 구강박테리아. 2023!!

[문형철]

• Review
• Open access
• Published: 11 October 2023

From nitrate to NO: potential effects of nitrate-reducing bacteria on systemic health and disease

• Hongyu Liu, 
• Yisheng Huang, 
• Mingshu Huang, 
• Min Wang, 
• Yue Ming, 
• Weixing Chen, 
• Yuanxin Chen, 
• Zhengming Tang & 
• Bo Jia 

European Journal of Medical Research volume 28, Article number: 425 (2023) Cite this article

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Abstract

Current research has described improving multisystem disease and organ function through dietary nitrate (DN) supplementation. They have provided some evidence that these floras with nitrate (NO3−) reductase are mediators of the underlying mechanism. Symbiotic bacteria with nitrate reductase activity (NRA) are found in the human digestive tract, including the mouth, esophagus and gastrointestinal tract (GT). Nitrate in food can be converted to nitrite under the tongue or in the stomach by these symbiotic bacteria. Then, nitrite is transformed to nitric oxide (NO) by non-enzymatic synthesis. NO is currently recognized as a potent bioactive agent with biological activities, such as vasodilation, regulation of cardiomyocyte function, neurotransmission, suppression of platelet agglutination, and prevention of vascular smooth muscle cell proliferation. NO also can be produced through the conventional l-arginine–NO synthase (l-NOS) pathway, whereas endogenous NO production by l-arginine is inhibited under hypoxia–ischemia or disease conditions. In contrast, exogenous NO3−/NO2−/NO activity is enhanced and becomes a practical supplemental pathway for NO in the body, playing an essential role in various physiological activities. Moreover, many diseases (such as metabolic or geriatric diseases) are primarily associated with disorders of endogenous NO synthesis, and NO generation from the exogenous NO3−/NO2−/NO route can partially alleviate the disease progression. The imbalance of NO in the body may be one of the potential mechanisms of disease development. Therefore, the impact of these floras with nitrate reductase on host systemic health through exogenous NO3−/NO2−/NO pathway production of NO or direct regulation of floras ecological balance is essential (e.g., regulation of body homeostasis, amelioration of diseases, etc.). This review summarizes the bacteria with nitrate reductase in humans, emphasizing the relationship between the metabolic processes of this microflora and host systemic health and disease. The potential effects of nitrate reduction bacteria on human health and disease were also highlighted in disease models from different human systems, including digestive, cardiovascular, endocrine, nervous, respiratory, and urinary systems, providing innovative ideas for future disease diagnosis and treatment based on nitrate reduction bacteria.

요약

현재의 연구에 따르면, 

식이성 질산염(DN) 보충제를 통해 

다중 시스템 질환과 장기 기능을 개선할 수 있다고 합니다. 

그들은 

질산염(NO3-) 환원효소를 가진 이러한 식물상이 

근본적인 메커니즘의 매개체라는 몇 가지 증거를 제시했습니다. 

질산염 환원효소 활성(NRA)을 가진 공생 박테리아는 

입, 식도, 위장관(GT)을 포함한 인간의 소화관에 존재합니다. 

Symbiotic bacteria with nitrate reductase activity (NRA)

음식에 함유된 질산염은 

이러한 공생 박테리아에 의해 

혀 밑이나 위에서 아질산염으로 전환될 수 있습니다. 

그런 다음, 

아질산염은 비효소 합성에 의해 산화질소(NO)로 전환됩니다. 

NO는 

현재 혈관 확장, 심근세포 기능 조절, 신경 전달, 혈소판 응집 억제, 혈관 평활근 세포 증식 억제 등 

생물학적 활성을 가진 강력한 생체 활성 물질로 알려져 있습니다. 

NO는 

기존의 l-arginine–NO synthase(l-NOS) 경로를 통해서도 생성될 수 있지만, 

저산소증이나 허혈 또는 질병 상태에서는 

l-arginine에 의한 

내인성 NO 생성이 억제됩니다. 

이와는 대조적으로, 

외인성 NO3−/NO2−/NO 활동은 강화되어 체내 NO를 보충하는 실질적인 경로가 되어 

다양한 생리 활동에 필수적인 역할을 합니다. 

또한, 많은 질병(대사성 질환이나 노인성 질환 등)은 주로 

내인성 NO 합성 장애와 관련이 있으며,

 외인성 NO3-/NO2-/NO 경로에서 NO가 생성되면 질병 진행을 부분적으로 완화할 수 있습니다. 

체내 NO의 불균형은 

질병 발생의 잠재적 메커니즘 중 하나일 수 있습니다. 

따라서, 

질산염 환원효소를 가진 이러한 식물상이 NO3-/NO2-/NO의 외인성 경로 생성을 통해 

숙주의 전신 건강에 미치는 영향 또는 

식물상 생태적 균형의 직접적인 조절(예: 신체 항상성 조절, 질병 개선 등)이 필수적입니다. 

이 리뷰는 

인간 내 질산염 환원효소를 가진 박테리아를 요약하여, 

이 미생물 군집의 대사 과정과 숙주의 전신 건강 및 질병 사이의 관계를 강조합니다. 

질산염 감소 박테리아가 인체 건강과 질병에 미치는 잠재적인 영향은 

소화기, 심혈관, 내분비, 신경, 호흡기, 비뇨기 계통을 포함한 

다양한 인체 시스템의 질병 모델에서도 강조되었으며, 

질산염 감소 박테리아를 기반으로 한 미래의 질병 진단 및 치료에 대한 혁신적인 아이디어를 제공합니다.

Introduction

With the increase in microbiological studies and advances in high-throughput sequencing technology in recent years, several publications on the contribution of microbiota in systemic health and the underlying mechanisms of action have emerged [1,2,3,4,5]. Among them, the bacteria with nitrate reductase are also gaining popularity among researchers [6], which can affect the systemic health and disease of the host by regulating nitrate metabolism [7] (including digestive system [8,9,10,11], cardiovascular system [12,13,14], endocrine system [15,16,17], nervous system [18,19,20], respiratory system [21,22,23] and urinary tract-related diseases [24,25,26]). There are symbiotic bacteria with NRA (such as micropore bacteria, actinomycetes, Escherichia coli, etc.) in the human oral cavity (OC), esophagus and GT, which are closely related to nitrate metabolism [11, 27,28,29,30]. Previous studies have shown that these symbiotic bacteria can reduce nitrate to nitrite, increasing nitrite concentration in plasma and saliva. Nitrite is further reduced to NO and exerts its biological activity after being swallowed into the GT [31]. Recent studies have described that DN supplementation can improve cognitive ability [21, 32, 33], skeletal muscle function [34, 35], cardiovascular function [36, 37] and other physiological functions closely related to human health [7, 38,39,40], and provide some evidence that the bacteria with nitrate reductase are the intermediary of the potential mechanism [41]. One of the mechanisms by which these floras affect human health is their involvement in the production of the signaling molecule NO, which is involved in most physiological activities in the human body (e.g., participation in metabolism and maintenance of cardiovascular homeostasis, dilation of blood vessels, inhibition of atherosclerotic angiopathy, etc.). Therefore, it can be inferred that bacteria with nitrate reductase in the human body are indispensable to human health and disease. The mechanism of its effect on human health deserves further study.

For more than 50 years, DN has been linked to the formation of nitrosamines and the development of cancer [42,43,44,45,46]. As a result, there are strict rules about acceptable levels of nitrate in our diet (e.g., food and drinking water, processed foods and cured meats). It has been observed that the lethal dose of oral nitrate in humans is about 330 mg/kg b.w., and the toxicity of sodium nitrite is about t times that of sodium nitrate. Dietary exposure estimates show that adults consuming 400 g of mixed vegetables does not exceed the daily intake of nitrate, which is within the daily intake range even considering nitrate exposure from other dietary sources. The acceptable daily intake (ADI) of nitrate determined by the former Food Science Council (SCF) was 3.7 mg/kg b.w./day, equivalent to 222 mg of nitrate per day for 60 kg adults and was reconfirmed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2002. At present, the human intake dose of nitrate and nitrite is within the safe range, far from the toxic dose of nitrate or nitrite [47]. In contrast, in the 1980s–1990s, numerous studies had shown that nitrate could not only biosynthesize in our bodies but also be reduced to NO and other closely related bioactive nitrogen oxides [48,49,50,51,52]. In recent years, it is believed that DN plays a powerful NO-like biological activity in human health [7].

This bioactivity is achieved through a series of reduction reactions. First, nitrite is formed by nitrate-reducing bacteria (NRB) and then transformed into NO and other bioactive nitrogen oxides by non-enzymatic synthesis [53]. With 80% of the body's nitrate coming from dietary leafy green vegetables, studies have shown that naturally occurring nitrate in vegetables is a beneficial active ingredient for systemic health compared to the nitrate added to processed foods and cured meats, and experts generally agree that supplementing nitrate with beetroot juice and other vegetable products may not be harmful [54]. DN is the precursor of signal molecule NO, and DN supplementation can improve NO bioavailability through the NO3−/NO2−/NO pathway [55]. NO is a mediator with biological activities, such as regulation of cardiomyocyte function [56], modulation of neurotransmission [57, 58], the principle of platelet function [59], anti-inflammation [60, 61], and prevention of vascular smooth muscle cell proliferation [62, 63]. Various studies demonstrated that supplementation of DN can diastole blood vessels [64], lower blood pressure (BP) [65], and improve oxygen consumption efficiency [66, 67]. One of the mechanisms of the effect of DN on human health is the production of NO through the NO3−/NO2−/NO pathway. Among them, NRB, which plays a vital role in nitrate metabolism, are colonized in the digestive tract (including the oral cavity, esophagus, and GT). Most of the nitrate in the human body is reduced in the OC. Therefore, most reports and studies on the effects of NRB in the human body on general health are biased towards oral floras [68, 69]. Considering the critical role of NO in human health, the body’s overall health may be intertwined with the existence of these bacteria. This review summarizes the relationship between the bacteria with nitrate reductase in human floras, the metabolic process, and the general health of the host. Through several disease models of different systems, the importance of bacteria with nitrate reductase in the human body to whole body health was highlighted to study and discover the methods and strategies to treat related diseases by interfering with the growth and metabolism of these NRB in the future.

소개

최근 몇 년 동안 미생물학 연구의 증가와 고처리 시퀀싱 기술의 발전으로 미생물 군집이 전신 건강에 기여하는 바와 그 작용 기전에 관한 여러 출판물이 등장했습니다 [1,2,3,4,5]. 그 중에서도 질산염 환원효소를 가진 박테리아는 질산염 대사를 조절함으로써 숙주의 전신 건강과 질병에 영향을 미칠 수 있는 것으로 연구자들 사이에서 인기를 얻고 있습니다[6]. (소화계[8,9,10,11], 심혈관계[12,13,14], 내분비계[15,16,17], 신경계[18,19,20], 호흡기 계통 [21,22,23] 및 요로 관련 질병 [24,25,26]).

인간의 구강(OC), 식도 및 GT에는

질산염 대사와 밀접한 관련이 있는 NRA(미세공극 박테리아, 방선균, 대장균 등)와

공생하는 박테리아가 있습니다 [11, 27,28,29,30].

micropore bacteria, actinomycetes, Escherichia coli

이전 연구에 따르면,

이 공생 박테리아는

질산염을 아질산염으로 환원시켜

혈장과 타액의 아질산염 농도를 증가시킵니다.

아질산염은

GT로 흡수된 후 NO로 환원되어

생물학적 활성을 발휘합니다 [31].

최근 연구에 따르면

DN 보충제는

인지 능력[21, 32, 33], 골격근 기능[34, 35], 심혈관 기능[36, 37] 및

인간의 건강과 밀접한 관련이 있는 기타 생리 기능[7, 38,39,40]을 향상시킬 수 있으며,

질산 환원 효소를 가진 박테리아가 잠재적 메커니즘의 매개체라는 증거를 제공합니다[41].

이러한 식물상이 인간의 건강에 영향을 미치는 메커니즘 중 하나는 인체 내 대부분의 생리 활동(예: 신진대사 참여, 심혈관 항상성 유지, 혈관 확장, 죽상경화성 혈관병증 억제 등)에 관여하는 신호 전달 분자 NO의 생성에 관여한다는 것입니다. 따라서 인체 내 질산염 환원효소를 가진 박테리아가 인간의 건강과 질병에 필수적이라고 추론할 수 있습니다. 이것이 인체 건강에 미치는 영향에 대한 메커니즘은 더 연구가 필요합니다.

50년 이상 동안, DN은 니트로사민의 형성 및 암 발생과 관련이 있었습니다 [42,43,44,45,46]. 그 결과, 식이요법에서 허용되는 질산염의 양에 대한 엄격한 규칙이 있습니다(예: 음식과 식수, 가공식품과 경화육). 사람의 경구 질산염 치사량은 약 330mg/kg b.w.이고, 아질산나트륨의 독성은 질산나트륨의 약 t배에 달하는 것으로 관찰되었습니다. 식이 노출 추정치에 따르면, 혼합 야채 400g을 섭취하는 성인의 경우, 다른 식이 공급원으로부터의 질산염 노출을 고려하더라도 일일 섭취량 범위 내에 있는 질산염 일일 섭취량을 초과하지 않는 것으로 나타났습니다. 구 식품과학위원회(SCF)가 규정한 질산염의 일일 허용 섭취량(ADI)은 3.7mg/kg b.w./day로, 이는 60kg 성인 기준 일일 222mg의 질산염에 해당하며, 2002년 식품첨가물에 관한 FAO/WHO 합동 전문가위원회(JECFA)에 의해 재확인되었습니다. 현재, 인체에 흡수되는 질산염과 아질산염의 양은 안전한 수준이며, 독성 수준과는 거리가 멉니다 [47]. 이와는 대조적으로, 1980년대와 1990년대에는 수많은 연구에서 질산염이 우리 몸에서 생합성될 수 있을 뿐 아니라 NO와 밀접하게 관련된 다른 생체 활성 질소 산화물로 환원될 수 있다는 사실이 밝혀졌습니다 [48,49,50,51,52]. 최근 몇 년 동안 DN은 인간의 건강에 강력한 NO와 유사한 생물학적 활성을 발휘하는 것으로 여겨지고 있습니다 [7].

이러한 생물학적 활성은 일련의 환원 반응을 통해 이루어집니다.

먼저, 질산염 환원 박테리아(NRB)에 의해 아질산염이 형성된 다음,

비효소 합성에 의해 NO 및 기타 생물학적 활성 질소 산화물로 변환됩니다 [53].

몸에 필요한 질산염의 80%가 잎이 많은 녹색 채소에서 나오기 때문에,

연구 결과에 따르면 가공식품과 경화육에 첨가된 질산염에 비해

채소에 자연적으로 존재하는 질산염은 전신 건강에 유익한 활성 성분이며,

전문가들은 일반적으로 비트 뿌리 주스 및 기타 채소 제품으로

질산염을 보충하는 것이 해롭지 않을 수 있다는 데 동의합니다 [54].

DN은 신호 분자 NO의 전구체이며,

DN 보충제는 NO3−/NO2−/NO 경로를 통해

NO 생체 이용률을 향상시킬 수 있습니다 [55].

NO는 심근세포 기능 조절 [56], 신경 전달 조절 [57, 58], 혈소판 기능 원리 [59], 항염증 [60, 61], 혈관 평활근 세포 증식 방지 [62, 63]와 같은 생물학적 활동을 하는 매개체입니다. 여러 연구에 따르면 DN 보충제는 혈관 이완을 촉진하고[64], 혈압을 낮추며[65], 산소 소비 효율을 향상시킵니다[66, 67]. DN이 인체 건강에 미치는 영향의 메커니즘 중 하나는 NO3−/NO2−/NO 경로를 통한 NO의 생성입니다. 그 중에서도 질산염 대사에 중요한 역할을 하는 NRB는 소화관(구강, 식도, GT 포함)에 서식합니다. 인체 내의 질산염 대부분은 구강에서 감소됩니다. 따라서 인체 내의 NRB가 전반적인 건강에 미치는 영향에 대한 대부분의 보고서와 연구는 구강 내 세균총에 편향되어 있습니다 [68, 69]. 인체 건강에 있어 NO의 중요한 역할을 고려할 때, 인체의 전반적인 건강은 이러한 박테리아의 존재와 밀접한 관련이 있을 수 있습니다. 이 리뷰는 인간 플로라의 질산염 환원 효소를 가진 박테리아, 대사 과정, 그리고 숙주의 전반적인 건강 사이의 관계를 요약합니다. 다양한 시스템의 여러 질병 모델을 통해, 미래에 이러한 NRB의 성장과 대사를 방해함으로써 관련 질병을 치료하는 방법과 전략을 연구하고 발견하기 위해, 인체 건강에 있어서 질산염 환원 효소를 가진 박테리아의 중요성이 강조되었습니다.

Relationship between NRB and their metabolic process and host's general health

NRB and DNNRB

NRB is a kind of bacteria with NRA. Nitrate is metabolized to nitrite by these symbiotic bacteria and further reduced to NO. This process has become essential to regulate NO homeostasis and signal transduction. There is no nitrate reductase in the human body, but there are symbiotic bacteria with NRA in the human digestive tract (including the OC, esophagus and GT) [11, 29, 31, 37, 69, 70] (Fig. 1). Therefore, NRB play an essential role in the NO3−/NO2−/NO pathway in intestinal salivary circulation.

NRB와 그들의 대사 과정 및 숙주의 전반적인 건강 상태와의 관계

NRB와 DNNRB

NRB는 NRA를 가진 일종의 박테리아입니다.

이 공생 박테리아에 의해

질산염이 아질산염으로 대사되고,

더 나아가 NO로 환원됩니다.

이 과정은 NO 항상성 및 신호 전달을 조절하는 데 필수적인 과정이 되었습니다.

인체에는 질산 환원효소가 없지만,

인간의 소화관(구강 점막, 식도, 위장 점막 포함)에는

NRA를 가진 공생 박테리아가 있습니다[11, 29, 31, 37, 69, 70] (그림 1).

따라서 NRB는 장내 타액 순환의 NO3−/NO2−/NO 경로에서 필수적인 역할을 합니다.

Fig. 1

Summary of NRB in the human OC, esophagus, and GT

Full size image

The microbial community of the OC consists of more than 700 prokaryotic taxa and 50–100 billion bacteria (including various NRB) [71,72,73]. NRB was first isolated from experimental rats in 1997 (including Staphylococcus minor, Staphylococcus intermedius, Pasteurella and Streptococcus). In addition, up to 65% of these bacteria are found deep in the posterior tongue [74]. Bacteria with nitrate reductase prefer an anaerobic environment; therefore, they are colonized in the deep fossa of the tongue. The most abundant species include Prevotella melanogaster, Heterotrichia, Haemophilus parainfluenzae, Neisseria flavus, Neisseria fines, and Clostridium nucleatum subsp. nucleatum, Campylobacter, C. labialis, and Prevotella intermedia [75,76,77,78]. Recently, some researchers have shown that the NRB in the human mouth mainly includes thick-walled bacteria (Staphylococcus, Streptococcus, Veronococcus), actinomycetes (Rosella, actinomycetes), Proteus (Neisseria, Haemophilus, campylobacter, Pasteurella), Bacteroides (Proteus) and so on [40]. Regardless of age, the most abundant group of NRB was micropore bacteria, and the level of NRB abundance in the mouth was positively correlated with the amount of DN supplement. The abundance of NRB would increase under a DN load [79].

A comparative analysis of the oral microbiota of vegetarians and omnivores by Hansen and colleagues [80] revealed that a higher proportion of Neisseria and Prevotella was associated with the intake of nitrate-rich vegetables. This suggests that a diet rich in nitrate increases the abundance of NRB and enhances the ability of oral floras to reduce nitrate to nitrite. In addition, the enzymatic activity of NRB varies in a bell-shaped pattern with age (peaking at 30–50 years). It may also vary according to the oral chemical environment (pH, saliva composition, periodontitis), diet type, hygiene practices, and gender [81]. More bacteria with NRA have been discovered in the OC, and the species are more and more abundant, indicating that NRB may play an indispensable role in human health. If NRB is deficient, the NO homeostasis in the body may be broken, and the health of the body will be affected to varying degrees. Tribble et al. [82] have demonstrated through 16S rRNA gene sequencing and analysis that healthy individuals with oral hygiene habits experienced a remarkable reduction in the diversity and abundance of bacteria with nitrate reductase in the mouth after using chlorhexidine mouthwash for a week (twice daily). The decrease in NRB led to a statistically considerable increase in systolic BP, an outcome confirmed in the previous reports [83]. In addition, NRB were also colonized in the oesophagus, which is directly connected to the OC, mainly by Lactobacillus spp., Streptococcus spp., Streptococcus spp., Actinomyces spp., and Prevotella spp. [84]. Some studies have shown that compared with the normal control group, the concentration of NRB in esophageal effusion samples of patients with non-progressive bulimia is significantly higher, especially in the Veronococcus spp., Lactobacillus spp. and Peptococcus spp. [85].

Gastric intestinal floras have been extensively studied in the last decade, especially the relationship between gastrointestinal floras and human health [86]. Bacteria with NRA (e.g., Streptococcus spp., Prevotella spp.) are also present in the GT [86,87,88,89]. Gastrointestinal NRB may have a role like that of the proven oral NRB involved in nitrate metabolism and further acting on the organism’s health. A nitrate-rich diet has recently been shown to increase the abundance of the faucal phylum bacteroides and reduce the quantity of the thick-walled phylum. This phenotype is associated with lower body weight and mass index [28]. Nevertheless, it has also been noted that 16S amplicon sequencing of faces collected from rats fed with high or low nitrate concentrations for 3 weeks found no differences in microbiome [90]. In addition, Rocha et al. [91] found that under antibiotic-induced bacterial symbiosis disorder, inorganic nitrate supplementation for 1 week could prevent the partial loss of rat faucal microflora, but there was no statistical difference. Does DN affect the metabolism of the gastrointestinal microflora? It has long been confirmed that DN enhances the ability of the GT to resist disease and infection, and antibiotic treatment that inhibits oral NRB increases the sensitivity of gastroenteritis. Nitrate in the diet can reverse the imbalance of gastrointestinal microflora caused by antibiotic therapy and enhance the defense of the GT [92, 93]. It is hypothesized that DN intake may modulate gastrointestinal flora metabolism and promote local redox-reduction interactions, thereby exerting beneficial effects on gastrointestinal flora and health status. Dysregulated NO metabolism is associated with ulcerative colitis (UC) [94]. NO can kill bacteria and regulate gastrointestinal mucosal blood flow (MBF) and mucus production, thus protecting the GT. Therefore, one of the mechanisms by which nitrate affects the GT is the role of NO produced by nitrate metabolism, and NRB takes an irreplaceable part in nitrate metabolism. Most DN is reduced to nitrite in the mouth through the enter salivary cycle. However, some nitrate enter the GT directly through the esophagus. There are more kinds of NRB in the OC, and most (about 80%) of the nitrate is reduced to nitrite in the OC through the intestinal saliva circulation, which is eventually catalyzed to generate other NO-related compounds. Most of them are absorbed by the stomach and upper small intestine before reaching the large intestine, and denitrification rarely occurs in the gastrointestinal tract, especially in the lower intestine [95].

Although it has been reported that long-term exposure to nitrate in food and drinking water is associated with an increased risk of colon cancer, its risk is limited to people with low vitamin C intake and high meat intake, suggesting that its risk may be affected by a combination of food and dietary nitrate [27, 96]. The formation of nitroso compounds (NOC) in the gastrointestinal tract is affected by a variety of environmental factors, including various nitrosation reagents, foods, gastric acid, and intestinal microflora [27]. Nitrate and nitrite in the diet come from vegetables and fruits. These foods contain many nitrosation inhibitors that can prevent cancer development [97]. In addition, European Food Safety Authority (EFSA) also pointed out in its 2008 report that epidemiological studies have not shown that nitrate intake from diet or drinking water is associated with an increased risk of cancer [47]. In summary, dietary intake of natural nitrate is almost impossible to denitrify in the lower intestine and is unlikely to produce carcinogenic NOC; the formation of NOC in the gastrointestinal tract is affected by a variety of environmental factors, and eating fruits and vegetables can inhibit the formation of NOC. Therefore, the current research mainly focuses on the effects of nitrate-reducing bacteria in the oral cavity on systemic health. These results further supported the concept that NRB affects human health through the enter salivary circulating NO3−/NO2−/NO pathway. Recently, studies on the effects of NRB on whole-body health have emerged, and the relationship between NRB and organismal fitness has become a focus of research.

OC의 미생물 군집은 700개가 넘는 원핵생물 분류군과 500억~1000억 개의 박테리아(다양한 NRB 포함)로 구성되어 있습니다 [71,72,73]. NRB는 1997년에 실험용 쥐에서 처음 분리되었습니다(Staphylococcus minor, Staphylococcus intermedius, Pasteurella, Streptococcus 포함).

또한, 이 박테리아의 최대 65%가 혀 뒤쪽 깊은 곳에서 발견됩니다[74].

질산 환원 효소를 가진 박테리아는

혐기성 환경을 선호하기 때문에 혀의 깊은 홈에 서식합니다.

가장 풍부한 종은 Prevotella melanogaster, Heterotrichia, Haemophilus parainfluenzae, Neisseria flavus, Neisseria fines, Clostridium nucleatum subsp. nucleatum, Campylobacter, C. labialis, Prevotella intermedia입니다 [75,76,77,78].

최근 몇몇 연구자들은

사람의 입안에 있는 NRB가 주로

두꺼운 벽을 가진 박테리아(Staphylococcus, Streptococcus, Veronococcus),

방선균(Rosella, actinomycetes),

프로테우스(Neisseria, Haemophilus, campylobacter, Pasteurella),

박테로이드(Proteus) 등을 포함하고 있다는 것을 밝혀냈습니다 [40].

연령에 관계없이 가장 풍부한 NRB 그룹은 미세공극 박테리아였으며,

입안의 NRB 풍부도는 DN 보충제의 양과 양의 상관관계가 있었습니다.

NRB의 풍부도는 DN 부하 상태에서 증가합니다 [79].

Hansen과 동료 연구진에 의한 채식주의자와 잡식주의자의 구강 미생물 군집 비교 분석 [80]에 따르면, 질산염이 풍부한 채소를 섭취하는 경우 Neisseria와 Prevotella의 비율이 더 높은 것으로 나타났습니다. 이것은 질산염이 풍부한 식단이 NRB의 양을 증가시키고 구강 미생물의 질산염을 아질산염으로 환원하는 능력을 향상시킨다는 것을 시사합니다.

또한, NRB의 효소 활성은 나이가 들면서 종 모양으로 변화합니다(30-50세에 최고치에 달함).

구강 내 화학 환경(pH, 타액 성분, 치주염),

식습관, 위생 습관, 성별에 따라 달라질 수도 있습니다 [81].

NRA를 가진 박테리아가 더 많이 발견되었으며,

그 종의 수가 점점 더 많아지고 있다는 것은

NRB가 인간의 건강에 없어서는 안 될 역할을 할 수 있음을 나타냅니다.

NRB가 부족하면 체내 NO 항상성이 깨질 수 있으며,

이로 인해 신체의 건강이 다양한 정도로 영향을 받을 수 있습니다.

Tribble et al. [82]은

16S rRNA 유전자 염기서열 분석과 분석을 통해

구강 위생 습관을 가진 건강한 사람이

클로르헥시딘 구강 세정제를 일주일 동안(하루에 두 번) 사용한 후

구강 내 질산 환원 효소를 가진 박테리아의 다양성과 수가 현저하게 감소하는 것을 확인했습니다.

NRB의 감소는

통계적으로 유의미한 수축기 혈압의 증가로 이어졌으며,

이는 이전 보고서에서 확인된 결과입니다 [83].

또한,

NRB는

주로 락토바실러스 종, 스트렙토코커스 종, 스트렙토코커스 종, 악티노미세스 종, 프레보텔라 종에 의해

OC에 직접 연결된 식도에 서식했습니다 [84].

일부 연구에 따르면, 비진행성 폭식증 환자의 식도 삼출물 샘플에서 NRB의 농도가 정상 대조군에 비해 유의하게 높으며, 특히 베로노코커스 종, 락토바실러스 종, 펩토코커스 종에서 더 높은 것으로 나타났습니다 [85].

위장 내 세균총은 지난 10년 동안 광범위하게 연구되어 왔으며, 특히 위장 내 세균총과 인간 건강의 관계에 대한 연구가 활발하게 이루어졌습니다 [86]. NRA를 가진 박테리아(예: Streptococcus spp., Prevotella spp.)도 GT에 존재합니다 [86,87,88,89]. 위장 내 NRB는 질산염 대사에 관여하고 유기체의 건강에 추가적인 영향을 미치는 것으로 입증된 구강 내 NRB와 같은 역할을 할 수 있습니다. 최근 질산염이 풍부한 식단이 구강 세균총의 박테로이드(bacteroides)의 양을 늘리고 두꺼운 벽을 가진 세균총의 양을 줄인다는 사실이 밝혀졌습니다. 이 표현형은 체중과 체질량 지수가 낮다는 것과 관련이 있습니다 [28]. 그럼에도 불구하고, 3주 동안 질산염 농도가 높거나 낮은 음식을 먹인 쥐의 얼굴에서 채취한 샘플을 16S 앰플리콘 시퀀싱을 통해 분석한 결과, 미생물 군집에 차이가 없다는 사실이 밝혀졌습니다 [90]. 또한 Rocha et al. [91]은 항생제 유발 세균 공생 장애 하에서 1주 동안 무기질 질산염을 보충하면 쥐의 구강 미생물총의 부분적 손실을 예방할 수 있다는 것을 발견했지만, 통계적 차이는 없었다.

DN은 위장 미생물총의 대사에 영향을 미치나요?

DN이 GT의 질병과 감염에 대한 저항력을 향상시킨다는 사실은

오랫동안 확인되어 왔으며,

구강 NRB를 억제하는 항생제 치료는

위장염의 민감성을 증가시킵니다.

식단에 질산염을 포함시키면

항생제 치료로 인한 위장 미생물 불균형을 회복하고

GT의 방어력을 강화할 수 있습니다 [92, 93].

DN 섭취가 위장 미생물 대사를 조절하고 국소적인 산화 환원 상호 작용을 촉진하여 위장 미생물과 건강 상태에 유익한 영향을 미칠 수 있다는 가설이 있습니다.

NO 대사 조절 장애는 궤양성 대장염(UC)과 관련이 있습니다 [94].

NO는 박테리아를 죽이고 위장 점막의 혈액순환(MBF)과 점액 생성을 조절하여 GT를 보호합니다. 따라서 질산염이 GT에 영향을 미치는 메커니즘 중 하나는 질산염 대사에 의해 생성되는 NO의 역할이며, NRB는 질산염 대사에서 대체할 수 없는 역할을 합니다. 대부분의 DN은 입안의 침 분비 순환을 통해 아질산염으로 환원됩니다. 그러나 일부 질산염은 식도를 통해 직접 GT로 들어갑니다. OC에는 NRB의 종류가 더 많으며, 대부분의 질산염(약 80%)은 장내 타액 순환을 통해 아질산염으로 환원되고, 이는 결국 다른 NO 관련 화합물을 생성하는 촉매 작용을 합니다. 이들 대부분은 대장에 도달하기 전에 위와 소장의 상부에서 흡수되며, 위장관, 특히 하부 소장에서 탈질화가 거의 발생하지 않습니다 [95].

음식과 식수 속의 질산염에 장기간 노출되면 대장암 발병 위험이 높아진다는 보고가 있지만, 그 위험은 비타민 C 섭취량이 적고 육류 섭취량이 많은 사람들에게만 국한된다는 점에서, 음식과 식이성 질산염의 조합이 위험에 영향을 미칠 수 있음을 시사합니다 [27, 96]. 위장관에서 니트로소 화합물(NOC)의 형성은 다양한 니트로화 시약, 음식, 위산, 장내 미생물 등 다양한 환경적 요인에 의해 영향을 받습니다 [27]. 식이요법에서 섭취하는 질산염과 아질산염은 채소와 과일에서 비롯됩니다. 이러한 식품에는 암 발생을 예방할 수 있는 많은 니트로화 억제제가 포함되어 있습니다 [97].

또한, 유럽식품안전청(EFSA)은 2008년 보고서에서 역학 연구 결과에 따르면 식이 또는 식수로부터의 질산염 섭취가 암 위험 증가와 관련이 있다는 사실이 밝혀지지 않았다고 지적했습니다 [47].

요약하면, 천연 질산염의 섭취는 하부 장에서 탈질화가 거의 불가능하며 발암성 NOC를 생성할 가능성이 낮습니다. 위장관에서 NOC의 형성은 다양한 환경적 요인에 의해 영향을 받으며, 과일과 채소를 섭취하면 NOC의 형성을 억제할 수 있습니다. 따라서 현재 연구의 주요 초점은 구강 내 질산염 환원 박테리아가 전신 건강에 미치는 영향에 집중되어 있습니다. 이 결과는 침의 순환 NO3-/NO2-/NO 경로에 의해 NRB가 인간의 건강에 영향을 미친다는 개념을 뒷받침합니다. 최근에는 NRB가 전신 건강에 미치는 영향에 대한 연구가 등장했고, NRB와 신체 건강의 관계가 연구의 초점이 되었습니다.

DN

Nitrate is widely present in water, soil, air, and plants. In addition, nitrate in humans is obtained from two primary sources: dietary intake (exogenous nitrate) and endogenous NO oxidation (endogenous nitrate) [98]. The human body takes nitrate from food, which accounts for about 80–85% of the total nitrate intake [99,100,101]. DN is primarily found in vegetables, including celery, radish, beet, etc. In addition, nitrate additives contained in meat are also part of the DN source [102]. Most of the DN is absorbed into the blood after entering the GT, and about 75% of the nitrate is excreted into the body in the form of urine through the kidney. About 25% of the remaining nitrate is enriched into the parotid gland with blood circulation and secreted into the mouth in the form of saliva, of which about 20% of the nitrate is reduced to nitrite under the action of NRB in the mouth [103,104,105]. The remaining nitrate and metabolic nitrite in saliva enter the stomach with swallowing and then enter a new round of the intestinal salivary circulation (80% of nitrite in the human body is produced by the intestinal salivary circulation) (Fig. 2); or NRB can reduce nitrate to nitrite in the acidic environment of the stomach. Then, nitrite is further reduced to biologically active NO and other biologically active nitrogen oxides by acid disproportionation, different hemoglobin (e.g., deoxyhemoglobin, deoxy myoglobin, neurohemoglobin, and cytoglobin), xanthine oxidase, proteins in the mitochondria (cytochrome c oxidase), and nitrite reductase. These nitrogen oxides can be signaled by nitrification, direct NO, and nitridation (Fig. 3) [98, 103, 106]. NO, a metabolite of DN, plays a vital role in protecting the cardiovascular system and gastrointestinal mucosa, regulating cardiomyocyte function and nerve transmission function, and playing a vital role in metabolic diseases. Symbiotic bacteria with nitrate reductase in the human body play an extremely significant role in the NO3−/NO2−/NO pathway, which reduces nitrate to nitrite. In addition, NO can be produced through the l-NOS path, a process regulated by NOS and its redox state [107].

Among them, NOS includes three subtypes: neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) [41, 108,109,110]. However, in the presence of hypoxia, ischemia, disease or ageing, NOS enzyme activity decreases, and endogenous NO production through the l-NOS pathway is reduced. Currently, the NO3−/NO2−/NO pathway activity is enhanced to maintain NO homeostasis [111, 112]. NO participates in numerous physiological functions of the human body (such as regulation of cardiomyocyte function, nerve transmission function, platelet function, etc.), so NO homeostasis is closely related to human health (Fig. 4). NO homeostasis is strongly associated with nitrate metabolism. NRB in the OC is essential in the process of nitrate metabolism [113, 114]. In addition, nitrate and nitrite are often used to suppress the development of microorganisms in meat products to prolong their shelf life. Nitrate and nitrite have been considered potential carcinogens for decades and are considered harmful [54, 115]. This stereotype has led to the neglect of the health benefits of nitrate. However, recent studies have failed to prove that nitrate harm humans [116,117,118]. The World Health Organization (WHO) issued guidelines on nitrate and nitrite in drinking water in 2011, arguing that nitrate are not carcinogens based on laboratory animal and epidemiological studies [119]. In addition, many studies have shown that dietary nitrate has many benefits to human health under the action of NRB (especially oral NRB).

DN

질산염은 물, 토양, 공기, 식물 등에 널리 존재합니다.

또한, 인간의 체내에서 질산염은

두 가지 주요 공급원으로부터 얻어집니다:

식이 섭취(외인성 질산염)와 내인성 NO 산화(내인성 질산염) [98].

인체는 음식에서 질산염을 얻는데,

이는 전체 질산염 섭취량의 약 80-85%를 차지합니다 [99,100,101].

식이 질산염(DN)은

주로 셀러리, 무, 비트 등을 포함한 채소에 함유되어 있습니다.

또한, 육류에 함유된 질산염 첨가물도 DN의 원천입니다 [102].

대부분의 식이질산염DN은 위장관GT에 들어간 후 혈액으로 흡수되며,

질산염의 약 75%는 신장을 통해 소변의 형태로 체외로 배출됩니다.

나머지 질산염의 약 25%는 혈액 순환을 통해 이하선에 축적되고,

타액의 형태로 입으로 분비되는데,

이 중 약 20%는 입안의 NRB의 작용으로 아질산염으로 환원됩니다 [103,104,105].

타액에 남아 있는 질산염과 대사성 아질산염은

삼킴을 통해 위장으로 들어가서 새로운 장내 타액 순환을 거칩니다

(인체 내 아질산염의 80%는 장내 타액 순환을 통해 생성됨)(그림 2).

또는

NRB가 위장의 산성 환경에서

질산염을 아질산염으로 환원할 수 있습니다.

그 다음, 아질산염은

산성 불균형,

다른 헤모글로빈(예: 데옥시헤모글로빈, 데옥시 미오글로빈, 신경 헤모글로빈, 사이토글로빈),

크산틴 산화효소,

미토콘드리아의 단백질(사이토크롬 c 산화효소),

아질산염 환원효소에 의해

생물학적으로 활성 NO와 다른 생물학적으로 활성 질소 산화물로 더 감소됩니다.

이러한 질소산화물은

질산화, 직접적인 NO, 질화 작용에 의해 신호가 전달될 수 있습니다(그림 3) [98, 103, 106].

DN의 대사 산물인 NO는

심혈관계와 위장 점막을 보호하고,

심근세포 기능과 신경 전달 기능을 조절하며,

대사성 질환에 중요한 역할을 하는 데 중요한 역할을 합니다.

인체 내 질산염 환원효소를 가진 공생 박테리아는

질산염을 아질산염으로 환원하는

NO3−/NO2−/NO 경로에서 매우 중요한 역할을 합니다.

또한,

NOS와 그 산화 환원 상태에 의해 조절되는

l-NOS 경로를 통해 NO가 생성될 수 있습니다 [107].

그 중에서도 NOS에는 세 가지 하위 유형이 있습니다:

신경질성 NOS(nNOS),

유도성 NOS(iNOS),

내피질성 NOS(eNOS) [41, 108,109,110].

그러나

저산소증, 허혈, 질병 또는 노화가 발생하면 NOS 효소 활동이 감소하고,

l-NOS 경로를 통한 내인성 NO 생산이 감소합니다.

현재,

NO3-/NO2-/NO 경로 활동이 강화되어

NO 항상성을 유지하고 있습니다 [111, 112].

NO는

인체의 수많은 생리 기능(심근세포 기능 조절, 신경 전달 기능, 혈소판 기능 등)에 관여하기 때문에,

NO 항상성은 인체 건강과 밀접한 관련이 있습니다(그림 4).

NO 항상성은

질산염 대사와 밀접한 관련이 있습니다.

OC의 NRB는 질산염 대사 과정에서 필수적입니다 [113, 114]. 또한, 질산염과 아질산염은 육류 제품의 미생물 발생을 억제하여 유통기한을 연장하는 데 자주 사용됩니다.

질산염과 아질산염은

수십 년 동안 잠재적인 발암 물질로 간주되어 왔으며

유해한 것으로 간주됩니다 [54, 115].

이러한 고정관념 때문에 질

산염의 건강상의 이점이 간과되었습니다.

그러나 최근의 연구들은

질산염이 인체에 해롭다는 것을 증명하지 못했습니다 [116,117,118].

세계보건기구(WHO)는 2011년에 질산염과 아질산염에 관한 지침을 발표하면서,

실험실 동물과 역학 연구에 근거하여 질산염은 발암 물질이 아니라고 주장했습니다 [119].

또한, 많은 연구에 따르면

식이성 질산염은 질산환원박테리아NRB(특히 경구 NRB)의 작용으로

인체 건강에 많은 이점을 가져다 줍니다.

Fig. 2

Metabolic process of nitrate in human body

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Fig. 3

Biochemical processes of nitrate metabolism

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Fig. 4

Bacteria with nitrate reductase ultimately reduce nitrate from dietary sources to NO via the enterosalivary cycle; the body can synthesize NO through the l-NOS pathway, but this pathway is inhibited by inflammatory factors or reactive oxygen species stimulation

질산염 환원효소를 가진 박테리아는 궁극적으로 식이성 질산염을 침-식도 순환을 통해 NO로 환원합니다. 신체는 l-NOS 경로를 통해 NO를 합성할 수 있지만, 이 경로는 염증성 인자 또는 활성 산소 자극에 의해 억제됩니다.

In addition, sialin is a membrane protein highly expressed in the parotid gland, a multifunctional anion transporter member of the SLC17 family [120]. Sialin is a salivary gland nitrate transporter with essential physiological effects in modulating NO3−/NO2−/NO homeostasis in the body (Fig. 5) [31]. Sialin was distributed in serous vesicle cells and lysosomal basal lateral membrane in the salivary gland [121]. The impaired function of the membrane protein sialin may have deleterious effects on human physiological functions [122]. Qin et al. [121] found proof of the physiological correlation of sialin in transporting circulating NO3− into the porcine salivary gland, and inhibition of sialin expression‎ reduced nitrate transport capacity. The discovery that the membrane protein sialin transports circulating nitrate into the parotid gland are critical for future research on the effects of NRB on human disease and health [123, 124].

또한, 시알린은 SLC17 계열의 다기능 음이온 수송체 구성원인 이하선에 많이 발현되는 막 단백질입니다 [120].

시아린은

타액선에서 NO3−/NO2−/NO 항상성을 조절하는 데 필수적인

생리적 효과를 가진 타액선 질산염 수송체입니다(그림 5) [31].

시아린은 타액선의 장액 소포 세포와 리소좀 기저 측면막에 분포되어 있습니다 [121].

막 단백질 시아린의 기능 장애는

인간의 생리적 기능에 해로운 영향을 미칠 수 있습니다 [122].

Qin et al. [121]은 순환하는 NO3−를 돼지 침샘으로 운반하는 데 있어서

시알린의 생리적 상관관계에 대한 증거를 발견했으며,

시알린 발현 억제가 질산염 운반 능력을 감소시켰습니다.

막 단백질인 시알린이 순환하는 질산염을 이하선에 운반한다는 사실은

NRB가 인간의 질병과 건강에 미치는 영향에 대한 향후 연구에 매우 중요합니다 [123, 124].

Fig. 5

Process of Sialin transporting nitrate in salivary gland cells

Effect of bacteria with nitrate reductase in the human microflora on systemic health

Many published studies show that DN is beneficial to human health, and NRB and NO play a significant part in the beneficial effects of nitrate. With the increase of age, or in some disease states, the pathway of classical enzymatic reaction to produce NO will be maladjusted. The NO3−/NO2−/NO path can compensate for the dysregulation of the classical way. Still, the NO3−/NO2−/NO pathway is disrupted when nitrate intake is inadequate, when antibacterial mouthwash/antibiotics are used, and when antacid therapy is used. These two pathways can compensate for each other, but when both systems fail, the NO-based signal will be suppressed entirely, eventually leading to disease. More studies are supporting the benefits of NRB on human whole-body health. From this review, we have discussed the influence of NRB on human systemic health through different disease models (Fig. 6).

인체 미생물 군집에 존재하는 질산염 환원효소를 가진 박테리아가 전신 건강에 미치는 영향

많은 연구 결과에 따르면,

DN은 인체 건강에 유익하며,

NRB와 NO는 질산염의 유익한 효과에 중요한 역할을 합니다.

나이가 들거나 일부 질병 상태에서는

NO를 생성하는 효소 반응 경로(NOS)가 제대로 작동하지 않게 됩니다.

NO3−/NO2−/NO 경로는

NOS에 의한 NO생성 방식의 조절 장애를 보상할 수 있습니다.

그러나

질산염 섭취가 불충분하거나

항균성 구강 세정제/항생제를 사용하거나

제산제 치료를 받는 경우,

NO3-/NO2-/NO 경로는 중단됩니다.

이 두 경로는 서로를 보완할 수 있지만,

두 시스템이 모두 실패하면

NO 기반 신호가 완전히 억제되어 결국 질병으로 이어집니다.

NRB가 인체의 전신 건강에 미치는 이점에 대한 연구가 더 많이 진행되고 있습니다.

이 리뷰를 통해 우리는 다양한 질병 모델을 통해 NRB가 인간의 전신 건강에 미치는 영향에 대해 논의했습니다(그림 6).

Fig. 6

Potential role of NRB in human systemic health and disease through the NO3−/NO2−/NO pathway

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Digestive system

Oral-related diseases

There is a lack of effective treatment for salivary gland dysfunction caused by radiotherapy for head and neck tumors (HNT). Feng et al. [125] discovered that nitrate supplementation in the diet effectively prevented radiation-induced parotid hypofunction in miniature pigs. Nitrate is a dose-dependent way to maintain the function of irradiated parotid gland tissue, thus preventing radiation-induced damage to the parotid gland. Nitrate addition to diet was shown to increase the expression‎ of sialin, a nitrate transporter, resulting in a positive feedback loop between nitrate and sialin. It can also stimulate the proliferation of human parotid cells (hPGCs) through the EGFR–AKT–MAPK signaling pathway. Furthermore, it is noteworthy that radiation treatment induces a hypoxic and acidic environment in the salivary gland. Under these circumstances, NO has been synthesized through the NO3−/NO2−/NO exogenous pathway, in which NRB is essential in reducing nitrate. Adding nitrate to the diet leads to the production of NO. It decreases hypoxia by inducing a long-term increase in blood flow and increasing glandular micro vascularization, facilitating salivary production by glandular vesicle cells [126]. Furthermore, nitrate-mediated NO generation can enhance the expression‎ of sialin and upregulate the EGFR–AKT–MAPK signaling pathway. This signaling pathway can promote cell proliferation, maintaining cell survival and preventing apoptosis [127, 128].

Therefore, the NO produced by DN through the NO3−/NO2−/NO pathway mediated by NRB may be a mechanism for avoiding xerostomia caused by radiotherapy. Recent studies have shown that radiotherapy and chemotherapy in patients with oral cancer (OCC) and oropharyngeal carcinoma (OPC) lead to oral ecological disorders and specific deletion of bacteria regulating the NO3−/NO2−/NO pathway in the intestinal salivary circulation [9]. Among them, Neisseria, Haemophilus, Porphyromonas, Clostridium and Cilium disappeared, while Lactobacillus increased, indicating that radiotherapy may lead to NO homeostasis imbalance. This also correlates with the significantly down-regulated oral metabolomic profile of NO-related precursors, regulators, or catalysts (e.g., aspartate, phenylalanine, l-ornithine, l-proline, xanthine, tyrosine, and glycine) in saliva samples from patients after radiotherapy. The decrease in the abundance of these NRB may lead to complications related to NO deficiency, such as xerostomia and local inflammation. Therefore, supplementation of NRB and DN in patients with head and neck radiotherapy and chemotherapy may be a new, safe, and effective method for treating radiation-induced xerostomia. We suggest that future studies explore the likelihood of oral microflora transplantation (OMT) and dietary interventions to reintroduce beneficial microorganisms in the OC of patients after head and neck radiation therapy to improve quality of life.

소화기관

구강 관련 질병

두경부 종양(HNT)에 대한 방사선 치료로 인한 침샘 기능 장애에 대한 효과적인 치료법이 부족합니다. Feng et al. [125]은 식이요법에서 질산염을 보충하면 미니어처 돼지에서 방사선으로 인한 이하선 기능 저하를 효과적으로 예방할 수 있다는 사실을 발견했습니다. 질산염은 방사선 조사된 이하선 조직의 기능을 유지하는 용량 의존적 방법이며, 따라서 방사선으로 인한 이하선 손상을 예방합니다. 식이에 질산염을 첨가하면 질산염 수송체인 시알린의 발현이 증가하여 질산염과 시알린 사이에 긍정적인 피드백 고리가 형성되는 것으로 나타났습니다. 또한, EGFR-AKT-MAPK 신호 전달 경로를 통해 인간 이하선 세포(hPGC)의 증식을 촉진할 수 있습니다.

또한,

방사선 치료가 침샘에 저산소 및 산성 환경을 유발한다는 점도 주목할 만합니다.

이러한 상황에서,

질산염은 NO3-/NO2-/NO 외인 경로를 통해 합성되는데,

이 과정에서 NRB는 질산염을 환원하는 데 필수적입니다.

식단에 질산염을 추가하면 NO가 생성됩니다.

NO는

혈류량을 장기간 증가시키고

선의 미세 혈관화를 증가시켜 저산소증을 감소시키고,

선포 세포에 의한 타액 생성을 촉진합니다 [126].

또한, 질산염 매개 NO 생성은 시알린의 발현을 강화하고

EGFR-AKT-MAPK 신호 전달 경로를 상향 조절할 수 있습니다.

이 신호 전달 경로는

세포 증식을 촉진하고,

세포 생존을 유지하며,

세포 사멸을 방지할 수 있습니다 [127, 128].

따라서,

NRB에 의해 매개되는 NO3-/NO2-/NO 경로를 통해

DN에 의해 생성된 NO는

방사선 치료로 인한 구강건조증을 피하는 메커니즘일 수 있습니다.

최근 연구에 따르면

구강암(OCC)과 구인두암(OPC) 환자의 방사선 치료와 화학 요법은

구강 생태계 장애를 유발하고

장 침 순환에서 NO3-/NO2-/NO 경로를 조절하는 박테리아의 특정 결실을 유발합니다 [9].

그 중 Neisseria, Haemophilus, Porphyromonas, Clostridium, Cilium은 사라지고, Lactobacillus는 증가하여 방사선 치료가 NO 항상성 불균형을 유발할 수 있음을 나타냅니다.

이것은 또한 방사선 치료 후 환자의 타액 샘플에서

NO 관련 전구체, 조절제 또는 촉매(예: 아스파르트산, 페닐알라닌, l-오르니틴, l-프롤린, 크산틴, 티로신, 글리신)의

구강 대사체 프로파일이 현저하게 감소하는 것과도 관련이 있습니다.

이러한 NRB의 풍부도가 감소하면 구강건조증 및 국소 염증과 같은 NO 결핍과 관련된 합병증이 발생할 수 있습니다. 따라서, 두경부 방사선 치료와 화학요법을 받는 환자들에게 NRB와 DN을 보충하는 것은 방사선으로 인한 구강건조증 치료에 있어 새롭고 안전하며 효과적인 방법이 될 수 있습니다. 우리는 향후 연구에서 구강 미생물 이식(OMT)과 식이 중재가 두경부 방사선 치료 후 환자의 구강 점막에 유익한 미생물을 다시 도입하여 삶의 질을 향상시킬 수 있는 가능성을 탐구할 것을 제안합니다.

Esophagus and GT-related diseases

Considerable evidence suggests that the microbiota has a crucial role in esophageal cancer [129, 130]. Li and his team [11] obtained matched pairs of saliva and esophageal brush samples from 276 subjects who underwent upper gastrointestinal endoscopy, using 16S rRNA analysis and next-generation sequencing technologies to study esophageal microbes. They found that compared with the normal group, the microbial diversity of saliva and cell brush samples decreased with the progression of the disease, and the nitrate reductase function of salivary floras in patients with esophageal squamous cell carcinoma (ESCC) decreased. It was also found that the part of nitrate reductase in matched esophageal brush samples from the same patient had the same change. The microflora in saliva and esophageal cancer cell brush samples are different, but the function of nitrate reductase is weakened, indicating that the bacteria with nitrate reductase can become a sensitive and specific clinical diagnostic marker for ESCC. In addition, it is suggested that NRB play a significant role in regulating the balance of esophageal microorganisms in patients with ESCC, which may be one of the critical mechanisms of the role of esophageal microorganisms in oesophagal cancer.

In addition to the esophagus, NRB can also be found in the stomach and may be closely associated with human health [86, 89, 131, 132]. A research team has, for the first time, published a report on the composition of gastric microflora during the development of gastric cancer. They found NRB (including Neisseria, Clostridium, and staphylococci) in the stomach and believed these bacteria were potential candidates associated with gastric cancer. However, the actual role of these bacteria in the development of gastric cancer has not been eval‎uated [86]. In addition, there is preliminary evidence that DN can protect the stomach by inducing gastric mucosal vasodilation, improving gastric mucosal blood flow, and promoting gastric mucus production. Recent studies suggest that these observations may be caused by NRB reducing nitrate through the NO3−/NO2−/NO pathway to produce NO in the human stomach [93, 133, 134]. Therefore, the specific effect of NRB on the stomach is worthy of our in-depth study.

DN can alter the gut microbial ecology system [75, 135, 136]. Hu and his colleagues [137] have demonstrated that DN supplements prevent colitis from upregulating carcinogenic pathways implicated in colorectal cancer development (e.g., activation of p53), indicating that nitrate may regulate inflammation by reshaping gut microbes. Previous studies have found reduced bacterial diversity and increased bacterial instability in patients with IBD compared to healthy individuals [138,139,140], confirm‎ing the changes in the microbiota of patients with IBD. However, the pathogenesis of IBD is still not fully understood, and a primary cause may be linked to the unbalance of intestinal bacteria. To investigate the mechanistic details leading to the dysbiosis of the intestinal bacteria in IBD, investigators assessed the role of nitrate in a mouse model of dextran sulfate sodium salt (DSS)-induced colitis [137]. The results revealed that nitrate addition to the diet maintained colonic consistency, improved colonic length, increased microvascular density, modulated serum Th cells, and decreased apoptosis rate in colonic epithelial cells, indicating that nitrate supplementation could reduce experimental colitis in mice [28, 137]. This suggests that nitrate can significantly ameliorate DSS-induced colitis by reducing the inflammatory response, decreasing apoptosis of colitis cells, improving intestinal blood flow, and activating the NO3−/NO2−/NO pathway to regulate intestinal floras [28, 141, 142]. Therefore, one of the pathogenic mechanisms of IBD may be that l-arginine-dependent endogenous NO synthesis is inhibited under inflammation, while exogenous dietary nitrate activates the NO3−/NO2−/NO pathway under the action of NRB to restore the balance of intestinal floras.

Cardiovascular system

Hypertension and pulmonary hypertension

Although we have improved the way we diagnose and image cardiovascular diseases (CD) at an early stage, with many new drugs approved for the treatment of CVD (such as hypertension, etc.), hypertension still puts us at risk of heart disease and stroke, which is the leading cause of death around the world [143]. Although the pathophysiological aspects of hypertension have been intensively studied in these decades, its incidence and preval‎ence have not decreased significantly. It is assessed that even with active drug therapy for hypertension, only approximately 50% of patients have their BP under control [25, 143]. At present, looking for new targets to prevent and treat hypertension is still the direction of our efforts. In 2006, the first study showed that inorganic nitrate reduced diastolic BP (3.7 mmHg) in healthy subjects 3 days after taking sodium nitrate (0.1 mmol/kg) [144]. Nitrate was later found to affect systolic blood pressure in a similar group of subjects [145,146,147]. A subsequent study validated previous claims that healthy volunteers reduced systolic and diastolic BP by 10.4 mmHg and 8 mmHg, respectively, after taking 500 ml of beetroot juice [147]. Interest in the effects of nitrate on cardiovascular function has been raised by these studies showing the potential of DN to lower BP. In recent years, it has been indicated that imbalances in the oral microbial community can negatively impact cardiovascular and metabolic health. Nitrate can produce NO in the human gastrointestinal tract through the NO3−/NO2−/NO pathway mediated by NRB, which may be one of the essential mechanisms of nitrate lowering BP in patients [12,13,14, 41, 148]. Recently, a review has elucidated the role of NO in the fight against CD, including hypertension, among others [149]. Evidence has shown that hypertension can be offset by drugs that improve NO signaling or restore NO bioavailability (for example, angiotensin-converting enzyme (ACE) inhibitors promote elevated levels of bradykinin, which activates the bradykinin B2 receptor in endothelial cells and eNOS, which increases NO production) [108]. Carlström et al. [150] further described the effects of dietary nitrate on various organ systems through the NO3−/NO2−/NO pathway under the action of NRB and discussed the potential mechanism of dietary inorganic nitrate reducing BP.

Small reductions in systolic BP in groups of people can remarkably decrease the risk of hypertension and mortality from CD (e.g., stroke). Therefore, exploring the effect of NRB on systemic blood pressure is significant. Evidence from studies has revealed that untreated hypercholesterolemic patients who consumed beetroot juice rich in nitrate for 6 weeks increased the abundance of NRB in the microflora of saliva while improving brachial flow-mediated dilation and reducing platelet monocyte aggregation [151]. More recently, Vanhatalo and his colleagues [14] showed in a clinical study that supplementation with nitrate-rich beet juice for 10 days similarly increased the abundance of NRB (Rhodobacter spp. and Neisseria spp.) in the salivary microbiota in both older (70–79 years) and younger (18–22 years) healthy subjects. They also found that acute (10 days) nitrate supplementation only reduced BP in healthy older adults, not in healthy younger adults. The current research results show that the oral microbial community is plastic and changes with the change of dietary inorganic nitrate intake, and the diet-induced changes in the oral microbial community are related to NO homeostasis and vascular health index. It has been shown that antimicrobial mouthwash reduces the number of NRB in the OC and can impair the antihypertensive and vasoprotective effects of l-arginine [152]. To explore the potential relationship between NRB and systemic BP, Petersson et al. [153] gave rats antibacterial mouthwash twice a day and supplemented nitrate drinking water simultaneously. They found that the simultaneous use of mouthwash significantly reduced the number of oral NRB compared with rats fed only nitrate drinking water, offsetting the drop in systemic BP caused by nitrate supplementation. More recently, data from various sets of animal models and humans have also revealed that antibacterial mouthwash can decrease the concentration of nitrite in the mouth and plasma while increasing BP by 2–3 mmHg [152,153,154,155,156]. Senkus et al. [83] performed an in-depth dissection of eight published studies between 2009 and 2016 (including five human crossover studies and three animal control studies). The data indicated that applying antibacterial mouthwash negatively affects saliva and plasma nitrate/nitrite concentrations, accompanied by increased BP. A review has elaborated that the disturbance of NO homeostasis by antibacterial mouthwash may cause an increased risk of cardiovascular mortality [53]. These findings demonstrated that DN supplementation increased the abundance of NRB, and that salivary nitrate can modulate cardiovascular function through the bioactivation of oral commensal bacteria. In contrast, overuse of antibacterial mouthwash may diminish the biological activity of DN. Goh et al. [78] pointed out that oral NRB can benefit the modulation of BP. Exploiting the NRA of specific symbiotic bacteria to make the NO3−/NO2−/NO pathway a potential system for maintaining NO bioavailability requires far-reaching and truly transformative research. Future longitudinal studies will strengthen the eval‎uation of the relationship between NRB exposure and hypertension, predict biomarkers of cardiometabolic risk and clinical disease progression, achieve early prevention of disease risk, and will provide information for the evolution of further intervention research manipulating oral NRB.

To explore the deeper mechanisms of the NO3−/NO2−/NO pathway to lower BP, Guimarães et al. [18] administered DN supplementation to angiotensin II (Ang II)-induced hypertensive rats and found that inorganic nitrate treatment not only reduced oxidative stress by promoting the NO3−/NO2−/NO pathway but also resulted in the suppression of sympathetic nerve activity (SNA) in this animal model or even normalized it, ultimately reducing BP in this animal model. They found that enhanced SNA boosts the progression of the disease, including hypertension, and raises the risk of adverse complications. A novel strategy to inhibit SNA may be of immense value in preventing or treating hypertension. Promoting the NO3−/NO2−/NO pathway through DN supplementation is a new nutritional and pharmacological approach to SNA inhibition in which NRB play an irreplaceable key role. However, this trial did not clarify that acute and chronic supplementation with inorganic nitrate can suppress SNA in hypertensive patients.

Recent studies have shown that S-nitrosation is impaired in hypertension, and increasing this modification may be an effective antihypertensive strategy. It is worth noting that NO plays a vital role in activating guanylate cyclase (GC), especially through the S-nitrosation of proteins. Nitrosylation affects G protein-coupled receptor (GPCR)-mediated signaling and can alter the affinity of angiotensin II for angiotensin II type 1 receptor and β-blocker transport [108]. These evidence suggests that this NO-mediated retro-translational modification may be closely related to vascular regulation and that NRB is a crucial contributor to NO production. Thus, NRB may regulate protein S-nitrosation through the NO3−/NO2−/NO pathway and thus effectively counteract hypertension.

In addition, it has been shown that when brassica vegetables rich in thioglucosides are consumed dietary (e.g., cauliflower and broccoli), mustard enzymes can convert thioglucosides to thiocyanate and increase serum thiocyanate levels [108]. Eating vegetables rich in nitrate can lower BP, but this effect will be eliminated when eating vegetables rich in nitrate and thiocyanate simultaneously. To investigate how this thiocyanate affects the antihypertensive effect of nitrate, Dewhurst-Trigg and his colleagues [157] found that consumption of thiocyanate-rich vegetables did not impact salivary nitrate intake but may inhibit the activity and metabolism of NRB, thereby affecting the capacity for nitrate conversion to nitrite in the OC. In addition, smoking increases the cycle level of thiocyanate, because cyanide in cigarette smoke is easily converted to thiocyanate through a desulphurization reaction catalysed by thiosulfate and 3-mercaptopyruvate. The study found that compared with non-smokers, smokers increased and decreased the concentration of nitrate in plasma and saliva while higher levels of thiocyanate in plasma and saliva. These phenomena are related to eliminating antihypertensive effects after smoking dietary nitrate in smokers. Both dietary intakes of thiocyanate-rich vegetables and smoking impair nitrate metabolism and the antihypertensive effect on this anion. It was recently demonstrated that smokers significantly impaired the conversion from nitrate to nitrite in the OC, suggesting that altered activity of oral NRB may be an essential pathophysiological mechanism.

Chronic gestational hypertension is significantly associated with poor outcomes in pregnancy, raising the risk of preeclampsia [158, 159], fetal growth restriction [160], and preterm birth. Dietary supplementation with nitrate has been shown to restore NO balance in the body, improve endothelial dysfunction, and lower BP. A clinical study has demonstrated a potential effect of DN (beet juice) supplementation on BP in hypertensive pregnant women [161]. The NO3−/NO2−/NO pathway process primarily involves the activity of bacterial nitrate reductase. It is hypothesized that variations in the effects of nitrate supplementation are associated with differences in individual microbiomes. Further studies must confirm‎ the relationship between chronic gestational hypertension and NRB. Future trials should explore and assess the beneficial influence of probiotic supplementation on the prognosis of pregnant women with hypertension. Probiotic interventions and supplementation with DN may suggest a safe and effective way to treat hypertensive disorders of pregnancy.

Moreover, pulmonary arterial hypertension (PAH) is a vascular disease in which mechanical obstruction increases in mean pulmonary arterial pressure [162]. Endothelial dysfunction, inflammatory and immune responses, and abnormal extracellular matrix function play a key role in PAH. PAH has vascular endothelial dysfunction and low NO bioavailability. Studies have shown that low-dose inhalation of NO (lasting for 4 h at 20 ppm concentration and then for 20 h at 6 ppm concentration) can improve oxygenation in PAH newborns without affecting the whole body and reduce systolic BP in nine PAH newborns [163, 164]. Subsequently, Roberts and his team [165] confirmed that inhaled NO increased systemic oxygen levels through a multicenter randomized controlled study of full-term and near-term infants with PAH. In addition to in vitro NO supplementation, restoration of NO bioavailability through dietary supplementation with NRB may be a dietary therapy for PAH.

As discussed earlier, the NRB-mediated NO3−/NO2−/NO pathway performs a vital role in preventing the progression of CD, for example, by reducing arterial stiffness, improving endothelial function, and reducing the risk of CD [12]. In addition, a recent study by feeding nitrate-added drinking water to mice with chronic ischemia in the hind limbs for 2 weeks found increased mobilization of CD34(+)/Flk-1(+) cells and migration of bone-marrow-derived (BMD)CD31(+)/CD45(−) cells to the site of ischemia, correlating with enhanced revascularization [166, 167]. These BMD endothelial progenitor cells (EPC) are activated in response to vascular stress and injury and are engaged in angiogenesis and restoration. The regenerative effect of DN can be abrogated using an antibacterial mouthwash, suggesting the importance of NRB in the NO3−/NO2−/NO pathway. Subsequently, the same acute mobilization of EPC was observed in the same studies after nitrate supplementation in humans [167,168,169]. These findings reveal that NRB may protect vascular function by mediating the NO3−/NO2−/NO pathway to affect the release and migration of various BMD cells.

However, due to the short duration and small sample size of the completed studies, the proof of the long-term effects of DN on BP in patients at increased cardiovascular risk is still not conclusive at this point. Therefore, future researchers need to use more accurate methods to eval‎uate the benefits and potential mechanisms of NRB in CD, such as hypertension, in clinical trials with a larger scale (> 300 participants) and longer DN supplementation time (> 12 months). The NO3−/NO2−/NO pathway determines NO homeostasis in cardiovascular health and disease. NRB plays a vital part in this pathway, providing a new target for the treatment of hypertension. We can modify CD patients’ prognosis through DN and probiotic supplementation. The findings of Goh et al. [78] support the role of oral NRB having a beneficial effect on BP modulation and insulin resistance (IR), which is one of the first studies to directly test the priori established hypothesis that oral NRB influences cardiometabolic outcomes. Future longitudinal studies will strengthen the estimation of the potential of NRB as biomarkers for predicting cardiometabolic risk and clinical disease progression and provide information for the evolution of future intervention studies that may manipulate oral nitrate-reducing capacity.

Heart disease

According to the most recent statistics on factors associated with heart disease reported annually by the American Heart Organs Association and the National Institutes of Health, diet is one-factor affecting heart health [170]. Nitrate is a potential dietary supplement for lowering BP. NRB acts as a key “driver” for lowering BP in patients through the NO3−/NO2−/NO pathway, with few studies investigating its effects on cardiac function. Under normal conditions, the decrease in BP due to vasodilation usually causes an increase in pressure reflex activity and heart rate, but with DN supplementation, only a drop in BP and no change in heart rate was observed [171, 172]. There are two explanations for this result: first, the effect of NO3−/NO2−/NO pathway mediated by NRB on BP is not enough to cause an increase in baroreflex activity and heart rate; second, NRB may have a direct or indirect inhibitory effect on baroreflex. Beetroot juice can be used as a natural nitrate supplement to increase the concentration of nitrite in plasma under the action of NRB [173]. In isolated and perfused Langendorff rat heart models, it has been shown that the increase of plasma nitrite concentration has a cGMP/PKG-dependent negative inotropic and muscular tone effect, characterized by a decrease in left ventricular relaxation and BP. This suggests that NRB may play an essential role in cardiac negative myodynamia and muscular tension.

In addition, studies have shown that the mechanisms of coronary dysfunction mainly involve inflammation, extensive and microvascular spasms, abnormal clotting and endothelial dysfunction [174, 175]. Endothelial dysfunction is a primary causative mechanism in patients with coronary heart disease risk factors. Endothelial dysfunction breaks down the endothelial l-NOS–NO pathway, contributing to a diminution in NO production, which leads to a decrease in endothelium-dependent diastolic effects and an increased risk of developing coronary artery disease, suggesting a critical function of NO in the regulation of endothelial function. Kanno et al. [176] similarly indicated that NO is vital in regulating cardiac function. They noted that in a healthy heart, NO produced by the endogenous NOS pathway reduces basal muscle force and plays a crucial role in safeguarding the myocardium from systolic/diastolic dysfunction, remodeling, and arrhythmias in the failing heart. Exogenous nitrate can produce NO through the NO3−/NO2−/NO pathway under the action of NRB, which is an independent alternative to the imbalance of endogenous pathways in a pathological state. Mechanisms by which NO acts include: (1) activation of soluble guanylate cyclase to produce cyclic guanosine monophosphate (cGMP) and relax vascular smooth muscle [177]; (2) increasing platelet cGMP levels to enhance anti-platelet agglutination and antithrombotic effects [178]; (3) decreases venous return and left ventricular end-diastolic pressure to diminish myocardial oxygen demand while increasing blood flow to the subendocardial; and (4) dilates coronary stenosis and increases collateral blood flow to directly increase myocardial oxygen supply [149, 179]. It is indicated that nitrate induces vasodilation of vascular smooth muscle through the NO3−/NO2−/NO pathway under the action of NRB, decreasing cardiac load and thus reducing left ventricular wall tension and end-diastolic pressure. It is a vital process in patients with stable ischemic heart disease (SIHD) as it not only reduces the load on the heart but also decreases the oxygen demand of the myocardium [180]. One study showed that nitrate could reduce myocardial ischemia and ischemic pain and increase exercise tolerance in stable and unstable angina pectoris. After acute myocardial infarction, nitrate can reduce ventricular dilatation, reducing pulmonary congestion and mitral regurgitation [181]. Besides, nitrate can also be used prophylactically before exercise, as they may improve exercise tolerance and avoid exercise-induced angina attacks [182, 183]. There is no doubt that nitrate have tremendous therapeutic potential in common cardiac diseases, such as angina pectoris. Currently, nitrate distributes coronary blood flow to ischemic areas through the NO3−/NO2−/NO pathway, thus improving local tissue hypoxia caused by illness as one of the many mechanisms to increase coronary perfusion. NRB, as the critical bacteria of nitrate metabolism to produce NO, few studies have clarified the potential mechanism of NRB in the recovery of coronary artery blood flow and reperfusion. The non-enzymatic and non-oxygen-dependent entero-salivary pathway is the primary mode of NO production, but an antibacterial mouthwash influences the effects produced by this pathway. A patient with angina pectoris reported in a case that had relief symptoms after discontinuation of antibacterial antiseptic mouthwash [184]. Therefore, we estimate that the NO3−/NO2−/NO path depends on the action of NRB to restore NO homeostasis and thus improve coronary perfusion.

Heart failure with reduced ejection fraction (HFrEF) is a fatal and disabling disease that is a significant public health concern. This disease is thought to be partially associated with the poor bioavailability of NO [181]. DN performs an essential part in treating conditions, including HFrEF, as a new source of human NO through intestinal salivary circulation and under the action of NRB. Some investigators have directly studied myocardial tissue from patients with HfrEF and found reduced cGMP levels, protein kinase G activity, and nitrite concentrations [185, 186]. Recent studies have shown that the impairment of coronary microvascular structure and function in HFpEF is primarily associated with decreased NO–cGMP bioavailability. When the organism is in a disease state, it leads to local microenvironmental hypoxia, which reduces endogenous NO production. We need new NO source pathways to restore NO homeostasis in the body [187,188,189]. The NO3−/NO2−/NO path is another strategy to enhance the NO–cGMP signaling pathway. Thus, NRB may serve as a suitable means to improve the efficiency of cardiac and peripheral NO signaling production at the early stages of the disease, thereby reducing the risk of disease progression to advanced settings. In addition, it has been shown that nitrite improves skeletal muscle mitochondrial efficiency, insulin sensitivity, and glucose uptake [190,191,192], so acute administration of inorganic nitrate treatment may enhance muscle strength in patients with HfrEF [192]. NRB is expected to be a probiotic to improve exercise capacity in patients with chronic HFpEF.

Endocrine systemII diabetes mellitus and metabolic syndrome

Type 2 diabetes (T2D) and metabolic syndrome (MS) are chronic non-communicable diseases with high preval‎ence and rapid growth rates worldwide [193, 194]. MS is like T2D in clinical signs and is also predominantly insulin resistant. Patients with MS may also have T2D, a major manifestation of disorders of the body’s metabolism of protein, fat, carbohydrate, and other substances [195]. A WHO report predicts that by 2040, the number of adults with T2D will exceed 650 million worldwide [15]. These diseases require long-term control and treatment, are costly to treat, and are correlated with an increased risk of death. An unhealthy diet (e.g., high in fat, sugar, etc.) is important in the increased incidence of T2D and MS, which are often associated with oxidative stress, impaired NO signaling, and CD [196, 197]. Oral microflora disorder is also considered to be related to the occurrence and development of T2D and MS [78, 198,199,200]. Among them, NRB, which have a significant part in the metabolic homeostasis of oral microorganisms, are of interest, because they are involved in a major part of NO production (NO3−/NO2−/NO pathway) [201], supplying an alternative systemic source of NO. NO is considered to be a signal molecule closely related to carbohydrate metabolism [202], so the imbalance of NRB in oral microorganisms is a risk factor for the impairment of carbohydrate metabolism and the occurrence and development of T2D and MS [78]. Under hyperglycemia, the impairment of the l-NOS–NO pathway leads to the decrease of NO synthesis and bioavailability. Some studies have shown that increased blood glucose and advanced glycation end products (AGEs) can down-regulate the expression‎/activity [203] of eNOS through inflammation [204], and redox pathway [205].T2D can lead to elevated cytokine levels (e.g., TNF-α) and further downregulate eNOS expression‎ [206]. This all leads to reduced endogenous NO synthesis and reduced bioavailability. In contrast, DN supplementation can compensate for the disturbance of the impaired enzyme-dependent pathway by promoting the NRB-dependent dietary NO3−/NO2−/NO pathway. Huang et al. [207] showed that mice deficient in eNOS evolve an MS-like phenotype with age. Carlstrom et al. [208] further showed that compared to controls, long-term dietary supplementation of nitrate decreased visceral fat accumulation, body weight gain, circulating triglycerides, and glycated hemoglobin (HbA1c) levels in mice and reversed the MS profile and prodromal diabetic phenotype in eNOS-deficient mice compared to controls. To investigate the relationship between T2D and MS and nitrate metabolism, Ohtake and his colleagues [209] found in a group of postmenopausal MS mouse models evoked by ovariectomy and a high-fat diet that these mice had lower circulating nitrate and nitrite levels compared to the corresponding controls and developed obesity, visceral adipocyte hypertrophy, and insulin resistance (IR), which would be avoided by nitrite treatment. Nyström and others [210] further pointed out that nitrite has dual stimulating effects on islet function, including indirect enhancement (increasing islet blood flow and redistribution through microcirculation) and direct insulin-promoting effect on β-cells. The insulin-promoting effect of nitrite is cGMP-dependent and involves the formation of active nitrogen and oxygen. In contrast, most nitrite in humans are produced by the reduction of DN by NRB. In addition, Khalifi et al. [211] investigated the effect of DN on glucose tolerance and lipids in a rat model of T2D induced by streptozotocin and nicotinamide injections. The results revealed that plasma nitrate and nitrite content decreased in these mice but recovered after nitrate supplementation, reducing hyperglycemia, and increasing blood lipid and glucose tolerance. Gheibi et al. [212] similarly found that obese T2D rats showed improved glucose tolerance, IR, and dyslipidemia after 2 months of DN supplementation compared to controls. These beneficial effects were correlated with increased GLUT4 expression‎ in insulin-sensitive tissues and reduced gluconeogenesis, inflammation, and oxidative stress. In addition, Li et al. [213] found that DN supplementation attenuated the elevation of circulating triglycerides, total cholesterol, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol induced by dietary interventions (high-fat and high-fructose diets). In summary, inorganic nitrate/nitrite supplementation showed good therapeutic effects in animal models of T2D and MS, as widely reported in other literature as [39, 213,214,215]. These anions increased insulin secretion from beta cells [210, 216] and improved peripheral glucose utilization [217, 218], visceral fat accumulation, and circulating levels of triglycerides. We hypothesize that endogenous NO deficiency may be responsible for MS and T2D and that NRB performs a critical part in the alternative pathway (NO3−/NO2−/NO pathway). Joshipua et al. [219] studied the effects of oral disinfectant rinses on more than 900 people over 3 years. The participants who used the mouthwash regularly were found to have a 55% higher risk of developing prediabetes/diabetes than those who used it infrequently. The authors did not examine the underlying mechanism of this process, but it may be that the use of antiseptic mouthwash blocked the action of NRB. In human clinical trials, nitrate and nitrite were ineffective in improving metabolic disorders [220,221,222]. Gilchrist and his colleagues [223] found that while dietary supplementation with nitrate caused a noticeable rise in circulating nitrite in patients with T2D, it did not improve islet function in patients with T2D. The cause of the paucity of effect was thought to be linked to the fact that T2D patients were being treated with bisphosphonates. The mechanisms of action of inorganic nitrate and biguanides share some striking similarities in many respects [224], leading to no significant effect of DN supplementation in patients already receiving biguanides. Among these, the interaction of nitrate and biguanides with the host microbiota may be central to the underlying mechanism [225,226,227]. Studies have shown that the high relative abundance of NRB in human OC is associated with insulin resistance and reduced risk of prediabetes [78]. Both animal and human trials have shown that alterations in the oral microbiota of patients with T2D lead to reduced nitrate reduction in the OC, decreased NO bioavailability, and the development of IR. In contrast, inorganic nitrate can modulate the oral microbiota by raising the number of health-related NRB and reducing the abundance of Prevotella and Weyongococcus spp., thereby increasing NO production and improving NO utilization. In addition, dietary epidemiological research has revealed that increased intake of nitrate-rich vegetables can convey weight loss and anti-diabetic effects and prevent the development of T2D [228, 229]. A review has discussed in detail how inorganic nitrate can improve the oral microbial community of patients with T2D and make it plays a probiotic role [230]. Further understanding of the pathophysiological mechanisms of T2D and MS will help develop new preventive and therapeutic strategies. Future animal studies or clinical trials will assess the potential beneficial effects of dietary supplementation with NRB in patients with metabolic diseases, including T2D. Combining the effects of the interaction of NRB with nitrate, restoring the oral microbiota of patients with T2D or MS to a state comparable to health is a state-of-the-art approach [41], which could enhance systemic NO production and offer an alternative way in the presence of impaired enzyme-dependent endogenous pathways. These findings may offer innovative nutritional prevention and treatment strategies for T2D and MS.

Nonalcoholic fatty liver

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, called hepatic steatosis or fatty liver, and is closely related to overweight, obesity, and MS [231, 232]. NAFLD can be reversed with weight loss and movement, which can also develop into severe diseases, including non-alcoholic steatohepatitis, fibrosis, and liver cirrhosis [233, 234]. Recent studies have shown that steatosis can be avoided by simple dietary approaches in rodent and human models of MS [209, 235]. In the latest trial, DN was proven to modify vascular function in patients with hypercholesterolemia [151]. Sonoda et al. [16] further found that high nitrate levels in diet positively affect liver steatosis associated with metabolic syndrome. DN provides fuel for the NO3−/NO2−/NO pathway, which can reverse many characteristics of metabolic syndrome and liver steatosis in high-fat diet mice (whether combined with NO synthase inhibitor (l-NAME) or not) [6, 236, 237]. Many scholars believe impaired NO bioavailability and signaling may be a candidate mechanism for hepatic steatosis [28, 238]. Lázár et al. [239] noted that the beneficial effects of nitrate metabolism could be attributed to its reduction of nitrite followed by further generation of NO species and activation of soluble guanylate cyclase [114, 240]. Furthermore, Cordero-Herrera et al.’s [15] in vivo disease model and in vitro studies using HepG2 cells and primary human hepatocyte spheres show that stimulating the NO3−/NO2−/NO pathway may slow down the evolution of hepatic steatosis by activating AMP-activated protein kinase (AMPK) signal transduction and reducing NOX-derived oxidative stress. These beneficial effects of nitrate were not present in germ-free mice, suggesting a central part of the host microbiota in the bioactivation of nitrate, which is necessary for DN to avoid steatosis. These findings may have implications for developing new strategies for the prevention and treatment of hepatic steatosis associated with metabolic disorders based on NRB.

Neuro system

Sympathetic excitation is accompanied by the increase of AngII signal and oxidative stress and impairs the bioavailability of NO [18, 19]. DN supplementation may influence the body's systemic health by modulating sympathetic activity and thus reducing vascular tone, regulating BP, and altering mental behaviors. These beneficial impacts of DN may be mediated through the inhibitory effect of the NO3−/NO2−/NO pathway on sympathetic excitation, in which NRB play an important role [18, 241]. Guimarães et al. [18] first demonstrated that long-term nitrate supplementation inhibited or restored regular sympathetic activity in an animal model of angiotensin-converting enzyme II-induced hypertension. However, this experiment did not directly verify the role of NRB but instead emphasized the significance of NRB in the regulation of NO homeostasis and signaling via the NO3−/NO2−/NO pathway. NO has a significant function in regulating synaptogenesis and neurotransmission in the central and peripheral nervous system [242]. There is evidence that basal NO production and NO bioavailability are reduced in diabetic patients [17]. Oghbaei et al. [243] used a ureazotocin-induced diabetic male rat model to demonstrate that long-term supplementation with DN can affect testicular function and structure in these rats through the hypothalamic–pituitary–gonadal axis, thereby improving fertility parameters. Subsequently, Keyhanmanesh et al. [244] also demonstrated the beneficial therapeutic effect of DN supplementation on testicular damage in streptozotocin-induced diabetic male rats. These findings are correlated with the increase in miR-34b and decrease in p53 mRNA, of which the role of NRB has not been revealed. We speculate that NRB may function as an essential part of this process. In addition, García-Jaramillo et al. [245] pointed out that DN supplementation altered the metabolomic profile of the zebrafish brain and led to mild anxiety-like behaviors. They suggest that DN supplementation can deplete brain metabolites (e.g., reduction of γ-aminobutyric acid and its precursor glutamine) by modulating neural activity, a process in which NO plays a vital role. We hypothesize that NRB is closely related to the operation of regulation of neural activity in humans, pending further experimental evidence. In addition to regulating nerve activity, DN can improve cognitive function by regulating blood flow to the brain and reducing response times in neuropsychological tests [246, 247]. Vanhatalo et al. [13] found that after DN supplementation, the microbiome module associated with pro-inflammatory metabolism decreased, and the microbial module with NRA increased and suggested that this microbial module alteration may be related to improvements in age-induced cognitive impairment. The relationship between NRB and cognitive function still needs further experimental validation.

Respiratory systemBronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is the most common severe respiratory complication in preterm infants and contributes significantly to the mortality of preterm infants [22, 248]. Currently, we lack treatments to block the progression of the disease as well as biomarkers to predict BPD. Despite the complexity of the aetiology and pathogenesis of BPD (which remains to be elucidated), recent studies demonstrated a relationship between the microecological environment of the digestive system (including the oral, respiratory, and intestinal tracts) and BPD. Among them, the correlation microbiome mediates NRA that regulates NO bioavailability and signaling, and defective NO bioavailability can lead to an increased incidence of BPD. Therefore, the bacteria with NRA in the OC may be the key to predicting and treating BPD [77, 249, 250]. In a single-center prospective cohort study, Gentle et al. [251] found that NRA peaked at 29 weeks of gestational age; when infants were categorized and compared by whether they had BPD, those with BPD had noticeably lower NRA at 29 weeks of gestational age than those without BPD. High NRA was associated with a lower incidence of BPD. This suggests that oral microflora and NRA may play a role in the occurrence of BPD in very premature infants. The difference in NRA mediated by oral microflora may provide a non-invasive biomarker for the development of BPD and has the potential for targeted therapy. In addition, studies have shown that significant changes in respiratory tract microecology (such as abnormal microbial diversity and differences in evolutionary patterns) have taken place in premature infants before the occurrence of BPD [252, 253]. Similarly, Wagner et al. [254] noted that the respiratory tract of infants with severe BPD has a higher weekly detectable microbial load than that of infants with mild BPD (with a lower colonization rate of Staphylococcus in the respiratory tract of infants with severe BPD). This suggests that the colonization pattern of respiratory microflora in premature infants may be a marker for predicting the severity of BPD. A review has elucidated in some detail the relationship between respiratory microbes and BPD and their primary mechanisms of action in the pathogenesis of BPD, suggesting that intervention of respiratory microecology with probiotics holds promise to become a new policy in the therapy of BPD [22]. Furthermore, there is growing evidence that the respiratory and gut microecological environments can interact, affecting the development of BPD. Dysregulation of the respiratory and gut microbiota can cause immune disorders and exacerbate the consequences of disease by stimulating inflammatory processes [255, 256]. Therefore, maintaining respiratory and intestinal microecological balance can significantly improve BPD. Studies have shown that there are also bacteria with NRA in the respiratory tract and intestinal tract. These bacteria are essential in regulating the microecological balance of the respiratory tract and intestinal tract [257, 258]. Therefore, we hypothesize that NRB may be highly promising targets for predicting and treating BPD. At present, NO and NO-producing precursors have been eval‎uated as potential BPD predictive markers and prevention methods, but the relationship between nitrate-reducing bacteria and BPD and the role of nitrate-reducing bacteria in predicting and treating BPD need to be further studied.

Urology systems

Chronic kidney disease

Chronic kidney disease (CKD) is a primary health problem affecting 8–16% of the global population [259]. Studies have shown that the microflora of patients with CKD is impaired (biological imbalance), increasing the number of potentially pathogenic and pro-inflammatory bacteria that can produce uremic toxins that facilitate the progression of CKD and decrease the production of enzymatic NO [24]. It has been confirmed in most animal experiments that the bioavailability of NO in patients with CKD is reduced. Since the decreased bioavailability of NO and increased oxidative stress are the key to the occurrence and development of renal disease [260], the therapeutic targets for NO may be beneficial [261,262,263]. In several disease models, DN supplementation has increased NO production and reduced endothelial dysfunction [145, 264]. In addition, supplementation with nitrite has been demonstrated to prevent or minimize renal damage directly by increasing NO production but has not been shown to improve the prognosis of patients with CKD [265, 266]. Similarly, we can also prevent or reduce renal impairment by increasing NO production through DN supplementation. Different ways of stimulating the NO3−/NO2−/NO pathway have been presented to avoid renal ischemia–reperfusion damage. Nitrate supplementation can improve renal injury in ischemia–reperfusion animal model [267, 268]. Two weeks of DN supplementation (1 mmol/kg/day) enhanced glomerular perfusion and filtration, prevented a decrease in glomerular filtration rate and attenuated glomerular and tubular injury in mice 2 weeks after ischemic renal injury [269]. Mechanistically, these renal protective effects associated with nitrate supplementation have been linked to reduced oxidative stress and increased NO bioavailability. A review detailed that the enhanced NO3−/NO2−/NO pathway may perform an antioxidant role through different targets and cellular mechanisms (including regulation of mitochondrial function, reduction of ROS produced by NOX and XO, and restoration of eNOS function) [25]. Although NRB is the only human symbiotic bacteria in the NO3−/NO2−/NO pathway, few studies clarify the relationship between NRB and CKD. As the impact of microorganisms on human health is gradually revealed, the microbiota can regulate the nitrogen cycle in various ways (e.g., through nitrification and denitrification, regulation of reduction and oxidation reactions, and through biological and chemical reactions) and play a crucial role in NO production [21, 270, 271]. One review has elaborated on how microorganisms regulate the nitrogen cycle [24]. Therefore, the effect of NRB on CKD may be a topic worth exploring in depth. In conclusion, the nitrogen cycle is an essential component of CKD prevention. Improper regulation of any part of the nitrogen cycle may promote the development of CKD. Regulating the nitrogen cycle by developing interventions that target NO production (e.g., supplementing NRB to regulate the microbiota) and thereby increasing NO production has the potential to reduce the risk of CKD. So far, few studies have fully described the role of NRB in the oral, esophageal or gastrointestinal microflora of patients with CKD. Therefore, we should focus on exploring this aspect in future studies and experiments, which may provide promising innovative ideas for predicting and treating CKD.

Summary and discussion

NRB perform a vital part in human health and diseases, because human beings lack the enzyme mechanism of reducing nitrate to nitrite, so we can only rely on NRB to reduce nitrate and make the NO3−/NO2−/NO pathway play a substitute role in the inactivation of the classical NOS pathway, thus restoring the balance of NO. In addition, NRB function as a critical regulator in the human floras. NRB have many beneficial effects on the body by restoring NO balance through the NO3−/NO2−/NO pathway or by regulating floras balance, including improving the hypofunction of salivary glands due to radiotherapy for HNT, regulating oral floras, protecting the stomach by improving gastric MBF and gastric mucus production, regulating intestinal floras, improving hypertension and pulmonary hypertension, alleviating heart disease, diabetes, MS, fatty liver, bronchopulmonary dysplasia, and chronic kidney disease. This review comprehensively summarizes the potential relationship between NRB and human health and conditions, an aspect that has received little attention and has not been summarized. NRB may be the key to the production of NO by nitrate metabolism and affect human health and may also be important bacteria for human health regulation. Our follow-up study can use these bacteria as a breakthrough to study the potential mechanism between nitrate metabolism, health, and disease. Therefore, this article accumulates a theoretical foundation for further studies on the effects of NRB on human health and diseases and stimulates more thoughts. The relationship between the function of microorganisms and the balance of human health and disease is becoming increasingly clear. Among them, studying the effects of bacteria with nitrate reductase on human systemic health and disease is still at an early stage. With the accelerated evolution of high-throughput sequencing and bioinformatics technologies, comprehensive microbiome characterization of NRB in different disease populations has become possible. According to the current research, the related studies on the relationship between NRB and human health and diseases are based on digestive tract floras (including the oral cavity, esophagus and gastrointestinal tract). This suggests a close relationship between NRB in our digestive tract and human health, which is worthy of in-depth excavation. Furthermore, NRB can influence the health of other body organs through the oral–intestinal axis, which can provide innovative ideas for disease diagnosis and treatment, such as finding biomarkers for specific diagnoses of diseases and treating-related conditions through probiotics by screening the changes of NRB in the digestive tract. In addition, the beneficial effect of NRB on the human body may be a protective mechanism of disease state or ageing, which not only indicates that NRB may be a potential therapeutic target but also may be a beneficial flora for improving age-related diseases, which needs to be further confirmed by more clinical trials. Recently, the modulation of the gut microbiota by NRB as beneficial commensals has attracted attention because of the potential effect of these floras on a variety of conditions and also as supplements to improve exercise performance [272, 273]. Using diet or supplements to change the microflora to increase the conversion of nitrate to nitrite, to increase the production and utilization of NO signal, which opens a new way in the field of sports performance. The use of high-throughput sequencing technology and biological information technology to understand the bacteria with nitrate reductase activity has been deepened, and a large number of data and information have been obtained. How to effectively transform big biological data into a clinical diagnosis and treatment method with practical application value and then provide effective personalized medical services for patients, there are still many problems to be solved. The growing development of metagenomics and high-throughput sequencing technologies significantly extended the human knowledge of the association between NRB and systemic diseases. Understanding the specific mechanism of NRB affecting human health and diseases and controlling NO3−/NO2−/NO metabolism is of great significance for preventing and treating human systemic diseases.

Availability of data and materials

This review does not involve statistics, and all the pictures are original.

References

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